Effects of water temperature on the lysosomal membrane stability in hemocytes of blacklip abalone, Haliotis rubra (leach).

ABSTRACT Neutral red retention (NRR) assay was used to evaluate the effect of changes in water temperature on lysosomal membrane integrity in the hemolymph of blacklip abalone, Haliotis rubra. Results from gradual temperature changes between 7[degrees]C and 16[degrees]C and between 16[degrees]C and 25[degrees]C showed that water temperatures within the range of 15[degrees]C to 17[degrees]C were optimal for maintaining lysosomal membrane integrity in this species. The rapid temperature changes between the ranges used in the gradual temperature change experiments indicated that when abalone were transferred directly between these temperatures their NRR time gradually increased or decreased to the level corresponding with the new temperature. However, when abalone were transferred directly between 7[degrees]C and 25[degrees]C or between 11.5[degrees]C and 20.5[degrees]C their NRR times initially decreased significantly, and then gradually increased to the levels corresponding with the new temperatures, indicating that different ranges of water temperature change can affect the lysosomal membrane integrity differently. The NRR times of blacklip abalone at 7[degrees]C, 16[degrees]C and 25[degrees]C were 40.0 [+ or -] 2.89 min, 113.33 [+ or -] 3.85 min and 35.0 [+ or -] 2.89 min, respectively.

KEY WORDS: neutral red retention assay, lysosomal membrane stability, temperature, blacklip abalone, Haliotis rubra

INTRODUCTION

The blacklip abalone, Haliotis rubra, is a marine mollusc found in the southern waters of Australia (Shepherd 1975). This temperate species predominantly inhabits rocky inshore waters between 5 and 30 m in depth (Shepherd 1975), where the water temperatures range from 8[degrees]C to 22[degrees]C (Gilroy & Edwards 1998, Shepherd & Hearn 1983). This species is also economically important, worth millions of export dollars a year to Australia (Drew et al. 2001). During the last decade blacklip abalone aquaculture has developed substantially and has become one of the main abalone species farmed in Tasmania and Victoria. Most commercial operations in Australia use land-based raceway systems in the grow-out phase, where the water temperatures can drop to 7[degrees]C in winter and increase to as high as 25[degrees]C in summer (A. Butterworth pers. comm.).

The published data showed that some abalone species had little tendency to adapt to chronically altered thermal environments and little ability to withstand acute thermal shock (Gilchrist 1995). For example, Prince et al. (1987) found that the presence of warmer water in the north and northeast of Tasmania was the main reason for the stunted growth of the wild blacklip abalone in the region. Unusual high water temperatures in summer were suggested by Winstanley (1972) as one of the major factors causing high abalone mortality in some regions of Australia. Correlations between high seawater temperature and increased mortality were also established in the experiments with the Californian black abalone H. cracherodii (Steinbeck et al. 1992). Cheng et al. (2004) noted that when the Taiwanese abalone (H. diversicolor supertexta) was subjected to elevated water temperatures they were more susceptible to Vibrio parahaemolyticus.

Lysosomes are polymorphic, hydrolytic enzyme-containing organelles with many intracellular and extracellular roles (De Duve & Wattiaux 1966). In molluscs, one of the main functions of lysosomes is to digest macromolecules and break down damaged or old cell parts as well as destroy foreign invaders such as bacteria and viruses (Hauton et al. 2001). Neutral red retention (NRR) assay measures the retention time of the neutral red dye in the lysosomes. In unstressed cells lysosomes will accumulate and retain the neutral red dye for an extended period of time. Once destabilized by a stress response the neutral red dye will leak into the cytosol of the cell through the damaged lysosomal membrane (Moore 1980, Pipe 1987, Lowe et al. 1995a, 1995b). As stressed cells lose their dye at a faster rate than nonstressed cells the rate of NRR in the lysosome can therefore be correlated to the overall stress of the animal (Harding et al. 2004a). Compared with other methods of measuring stress in molluscs NRR assay is convenient and inexpensive, thus this methodology is increasingly being applied as an index to evaluate environmental, physiological and mechanical stresses (Hauton et al. 1998, 2001). NRR assay has been used in assessing responses to various contaminants in mussels and freshwater snails (Lowe & Pipe 1994, Lowe et al. 1995a, b, Mamaca et al. 2005, Svendsen & Weeks 1994), to seasonal and environmental changes associated with the reproductive cycle, temperature, air exposure and food availability in mussels and oysters (Harding et al. 2004b, Zhang et al. 2006) and to mechanical disturbances related to grading, postharvest processing activities and storage conditions in mussels and oysters (Harding et al. 2004a, Zhang & Li 2006).

The main objective of this study is to investigate if NRR assay could be used to evaluate the effects of water temperature change on blacklip abalone H. rubra and their responding patterns in controlled experiments.

MATERIALS AND METHODS

Experimental Animals

The abalone used in this study were obtained from a commercial farm located in Victoria and were approximately four years of age. Their height, length and width (mean [+ or -] SE, n = 100) were 29.15 [+ or -] 0.60 mm, 93.71 [+ or -] 1.16 mm and 71.94 [+ or -] 0.89 nun, respectively. During the experimental period the abalone were held in 500 L tanks and fed on a standard artificial abalone diet provided by Adam & Amos Abalone Diets (Mt Barker, Adelaide, South Australia).

Neutral Red Retention Assay

The Neutral red retention (NRR) assay used in this study was modified from the methods used by Lowe et al. (1995a, 1995b), Hauton et al. (1998, 2001) and Zhang et al. (2006). The neutral red stock solution was made by dissolving 2.28 mg of neutral red powder in 1 mL of dimethyl sulphoxide (DMSO). The working solution (0.01 mg [mL.sup.-1]) was prepared by diluting 17 [micro]L of the stock solution with 4 mL of artificial saline solution consisting of 0.48 g [L.sup.-1] Ca[Cl.sub.2], 1.45 g [L.sup.-1] MgS[O.sub.4], 2.18 g [L.sup.-1] Mg[Cl.sub.2] x 6[H.sub.2]O, 0.31 g [L.sup.-1] KCl, 11.61 g [L.sup.-1] NaCl, and 0.35 g [L.sup.-1] NaHC[O.sub.3] (Buchanan et al. 2001).

Abalone hemolymph (0.2 mL) was drawn from the cephalic arterial sinus at the anterior end of the foot using a 29 G x 1-mL syringe. The hemolymph was then placed into a 2 mL siliconized Eppendorf tube containing 0.2 mL of artificial saline solution and gently mixed. Each abalone hemolymph sample was collected within 1 min to minimize the potential effects from handling. A 40-[micro]L mixture of the hemolymph sample and artificial saline solution was then placed onto a microscope slide treated with a poly-L-lysine solution (20 [micro]L poly-L- lysine in 100 [micro]L distilled water). The slide was then placed into a 10[degrees]C lightproof humidity chamber for 15 min to allow the cells to adhere to the slide. After 15 min the slide was removed from the chamber and the excess hemolymph was removed, 20 [micro]L of neutral red working solution (10[degrees]C) was then added to the cell layer and incubated in the chamber for another 15 min. A cover slide (22 x 22 mm) was then placed onto the slide and the hemocytes were examined using a compound microscope (x400 magnification). The slide was examined at 15 min intervals for the first 60 min and then every 20 min. A total of 50 granulocytes per individual were examined at each time interval. Once 50% of the hemocytes had started to lose dye from their lysosomes the assay was stopped and the time for the previous examination was recorded as the NRR time. The observer was not informed of the origin of the sample under examination to minimize the possibility of biased assessment.

The Effect of Water Temperature Change on Lysosomal Membrane Stability

In this study each temperature treatment group was replicated three times by using three 500-L flow through tanks. Prior to each treatment 60 tagged abalone per replicate were acclimated at the required starting temperatures for seven days. At each sampling time hemolymph was taken from three randomly selected abalone in each replicate, with each of these abalone sampled once only.

Gradual Water Temperature Change

The gradual water temperature change experiment consisted of two treatments. In the first treatment, abalone acclimated at 16[degrees]C were split into three groups. The first two groups were respectively subjected to a gradual increase or decrease in water temperatures, whereas the third group was maintained at 16[degrees]C as a control. A temperature change rate of 1[degrees]C every two days was applied in the first two days and was changed to 2[degrees]C every two days thereafter until the final temperature of 7[degrees]C or 25[degrees]C was reached. In the second treatment abalone were also separated into three groups. In the first group abalone acclimated at 7[degrees]C were subjected to a gradual increase, whereas in the second group abalone acclimated at 25[degrees]C were subjected to a gradual decrease in water temperature until the final temperature of 16[degrees]C was reached in both groups. A temperature change rate of 2[degrees]C every two days was used until 15[degrees]C or 17[degrees]C was reached and then changed to 1[degrees]C every two days to the final temperature of 16[degrees]C. The third group was maintained at 16[degrees]C as a control. Hemolymph was sampled 48 h after the required temperature change. Once the final temperatures were reached the abalone were maintained for a further 6 days with hemolymph samples taken every 48 h during this period. The control samples were collected at each sampling time from the 16[degrees]C tanks.

Rapid Water Temperature Change

The rapid temperature change experiment consisted of four treatments. In the first treatment, abalone acclimated in 16[degrees]C water were split into two groups and transferred into 7[degrees]C and 25[degrees]C water respectively. In the second treatment two groups of abalone were acclimated at 7[degrees]C and 25[degrees]C respectively and then transferred directly into 16[degrees]C water. In the third treatment the first group was acclimated at 7[degrees]C and placed directly into 25[degrees]C water, whereas the second was acclimated at 25[degrees]C and placed into 7[degrees]C water. In the fourth treatment the abalone acclimated at 11.5[degrees]C were placed directly into 20.5[degrees]C water, whereas those acclimated at 20.5[degrees]C were placed directly into 11.5[degrees]C water. In each treatment the hemolymph was sampled at times 0, 0.5 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h and on days 5 and 7, respectively.

Statistical Analysis

All statistics were run with SPSS 13.0 software. The normality test was conducted first and showed normal distribution on all the data used in this study. ANOVA and Tukey b multiple comparisons were then applied. A probability level of P < 0.05 was considered statistically significant.

RESULTS

Gradual Water Temperature Change

The results from the first treatment indicated that a gradual decrease or increase in water temperature from 16[degrees]C produced a declining trend in NRR time. When the water temperature was decreased from 16[degrees]C to 7[degrees]C (Fig. 1A) the NRR time did not change significantly (P > 0.05) between 16[degrees]C and 15[degrees]C. The NRR time then dropped significantly (P < 0.05) to 77.78 [+ or -] 8.01 min at 13[degrees]C, and further decreased to 33.33 [+ or -] 3.33 min on day 10 at 7[degrees]C. The NRR time then fluctuated at this level until the end of the experiment. When the water temperature was gradually raised from 16[degrees]C to 25[degrees]C (Fig. 1B) the NRR time did not change significantly (P > 0.05) between 16[degrees]C and 17[degrees]C. Further water temperature increase from 17[degrees]C to 19[degrees]C produced a significant decrease in NRR time (P < 0.05), dropping from 108.89 [+ or -] 5.88 min to 76.11 [+ or -] 5.47 min. The NRR time declined to 31.67 [+ or -] 1.67 min on day 10 at 25[degrees]C and then remained at this level until the end of the experiment.

[FIGURE 1 OMITTED]

In the second treatment the gradual water temperature change from 7[degrees]C or 25[degrees]C to 16[degrees]C produced an increasing trend in NRR time. When water temperature was increased from 7[degrees]C to 16[degrees]C (Fig. 2A) the lowest NRR time of 40.0 [+ or -] 2.89 min was observed on Day 0 at 7[degrees]C. The NRR time then increased gradually as the temperature was increased over time. At the temperature of 15[degrees]C on day 8 the NRR time reached a level that was not significantly different from the control (P = 0.352). The NRR time increased slightly on day 10 once the temperature was changed to 16[degrees]C and remained at this level until the end of the experiment. When water temperature was decreased from 25[degrees]C to 16[degrees]C (Fig. 2B) the lowest NRR time of 35.0 [+ or -] 2.89 min was observed on day 0 at the temperature of 25[degrees]C. The NRR time then increased gradually as the temperature was decreased over time. On day 8 the NRR time was similar to the control (P = 0.848) when 17[degrees]C was reached. The NRR time further increased slightly at 16[degrees]C and remained at this level until the end of the experiment.

[FIGURE 2 OMITTED]

During the experimental periods no significant difference in NRR times occurred in the controls in the two treatments (P > 0.05).

Rapid Water Temperature Change

In the first treatment when blacklip abalone were transferred directly from 16[degrees]C to 7[degrees]C or 25[degrees]C water a rapid decline in NRR time was produced (Fig. 3). The NRR time dropped significantly (P < 0.05) at each sampling time point within the first three to six hours from the initial level of 113.33 [+ or -] 3.85 min at 0 h to 50.0 [+ or -] 2.89 min at 3 h in the 16[degrees]C to 7[degrees]C group and to 41.67 [+ or -] 1.67 min at 6 h in the 16[degrees]C to 25[degrees]C group. The NRR time then decreased slightly to 36.67 [+ or -] 3.33 min and 31.67 [+ or -] 1.67 at 12 h in these two groups respectively. Statistical analysis showed that from 6 h to the end of the experiment the NRR times in each group did not differ significantly (P > 0.05).

[FIGURE 3 OMITTED]

In the second treatment when abalone were transferred from 7[degrees]C or 25[degrees]C to 16[degrees]C the NRR times dropped from the initial levels of 40.0 [+ or -] 2.89 min and 35.0 [+ or -] 2.89 min at 0 h to 30.0 [+ or -] 2.89 min and 31.67 [+ or -] 1.67 min at 0.5 h, respectively (Fig. 4). From 0.5 h onwards the NRR time gradually increased over time. By 12 h the NRR times for the 7[degrees]C to 16[degrees]C and 25[degrees]C to 16[degrees]C groups were 93.33 [+ or -] 3.85 min and 93.33 [+ or -] 3.85 min, respectively, which was not significantly different (P > 0.05) from the controls.

[FIGURE 4 OMITTED]

During the experimental periods no significant differences in NRR time occurred in the controls for the first two treatments respectively (P > 0.05).

In the third treatment, when the abalone were transferred directly from 7[degrees]C to 25[degrees]C the NRR time dropped significantly (P = 0.014) within the first 3 h from the initial level of 40.0 [+ or -] 2.89 min at 0 h to 20.0 [+ or -] 2.88 min at 3 h and decreased further to 16.67 [+ or -] 1.67 min at 12 h. From 12 h onwards the NRR time gradually increased to 31.67 [+ or -] 1.67 min on day 7 (Fig. 5). When abalone were transferred directly from 25[degrees]C to 7[degrees]C the NRR time also dropped significantly (P = 0.022) within the first 6 h from the initial level of 35.0 [+ or -] 2.88 min at 0 h to the lowest level of 18.33 [+ or -] 1.67 min at 6 h. From 6 h onwards the NRR time gradually increased to 31.67 [+ or -] 4.41 min at 48 h and then fluctuated around this level until the end of the experiment. The mortality levels of the direct temperature changes from 7[degrees]C to 25[degrees]C and 25[degrees]C to 7[degrees]C were 24.6% and 17.5% respectively.

[FIGURE 5 OMITTED]

In the fourth treatment it was observed that the 11.5[degrees]C to 20.5[degrees]C temperature change group produced a significant drop (P < 0.05) in NRR time from the initial level of 77.78 [+ or -] 5.88 min at 0 h to 38.33 [+ or -] 4.41 min at 1.5 h. The NRR time then increased gradually over the remaining sampling times, reaching 73.33 [+ or -] 3.85 min on day 7 (Fig. 6). In the 20.5[degrees]C to 11.5[degrees]C temperature change group the NRR time dropped significantly (P < 0.05) within the first 0.5 h from the initial level of 73.33 [+ or -] 3.85 min at 0 h to 38.33 [+ or -] 1.67 min at 0.5 h. From 1.5 h onwards the NRR time increased gradually and reached 66.67 [+ or -] 3.85 min on day 7 (Fig. 6). No mortalities were recorded in this treatment.

[FIGURE 6 OMITTED]

The Effect of Rapid Versus Gradual Temperature Change on Lysosomal Stability at Final Temperature

Analysis of NRR times at 7[degrees]C, 16[degrees]C and 25[degrees]C indicated that the lysosomal membrane stabilities at these temperatures were not significantly affected by the speed (gradual or rapid) of change of water temperature (P > 0.05).

DISCUSSION

When water temperature was changed by 1[degrees]C from 16[degrees]C in the gradual temperature change experiments the NRR times changed slightly (P > 0.05). As water temperature was further changed to lower than 15[degrees]C or higher than 17[degrees]C a significant drop in the NRR times was produced. These results indicate that temperatures between 15[degrees]C and 17[degrees]C are optimal for maintaining lysosomal membrane stability in blacklip abalone H. rubra. These results are also consistent with the findings in the published studies on this species by using performance and growth rate as indicators. Gilroy and Edwards (1998) found that the preferred temperature for H. rubra was 16.9[degrees]C and the optimum temperature for their growth was 17[degrees]C, which was supported by the finding of Harris et al., in 2005 that the highest recorded growth for H. rubra was at the temperature of 17[degrees]C. Harris et al. (2005) also noted that at the temperature of 19[degrees]C the growth rate of H. rubra was reduced considerably.

The rapid temperature change experiment showed that when abalone were transferred directly from 16[degrees]C to 7[degrees]C or 25[degrees]C water the NRR times dropped to the levels that were not significantly different from those corresponding with 7[degrees]C or 25[degrees]C temperature within 3-6 h. When abalone were transferred from 7[degrees]C or 25[degrees]C to 16[degrees]C water their NRR times increased to the optimal level within 12 h. These results indicate that when H. rubra is subjected to rapid water temperature changes the lysosomal membrane destabilization is quicker than its stabilization. Similar results were also found by Zhang et al. (2006) with the Pacific oyster, Crassostrea gigas. In their experiments, when oysters were transferred from 15[degrees]C to 5[degrees]C or 25[degrees]C water, the NRR time decreased to the levels corresponding with 5[degrees]C or 25[degrees]C water within 3 h, which was substantially less than the five days required in the reverse experiment in which C. gigas was transferred from 5[degrees]C or 25[degrees]C to 15[degrees]C.

In the gradual and rapid temperature changes between 7[degrees]C and 16[degrees]C and between 16[degrees]C and 25[degrees]C the lowest NRR times were recorded at 7[degrees]C and 25[degrees]C, suggesting that these temperatures produced the greatest impact on lysosomal stability. However, no mortalities were recorded in these treatments, indicating that H. rubra could tolerate temperature changes in these ranges.

When blacklip abalone were subjected to the direct water temperature changes between 7[degrees]C and 25[degrees]C or 11.5[degrees]C and 20.5[degrees]C the NRR time decreased to the levels significantly lower than those previously recorded for these temperatures at the acclimation stages. It was also noted that after an initial decrease the NRR time increased to the levels corresponding with 7[degrees]C, 11.5[degrees]C, 20.5[degrees]C or 25[degrees]C water gradually, indicating that the lysosomal membrane had the ability to recover from this extra stress. In the between 7[degrees]C and 25[degrees]C treatments, a mortality level of more than 17.5% and a slower recovery in NRR times were recorded, suggesting that these temperature changes had produced the strongest physiological impact on the abalone in this study.

The differences between the start and the end temperatures were all 9[degrees]C in the rapid temperature changes between 7[degrees]C and 16[degrees]C, 16[degrees]C and 25[degrees]C and 11.5[degrees]C and 20.5[degrees]C. However, only the between 11.5[degrees]C and 20.5[degrees]C treatments produced the extra stress response, suggesting that different ranges of water temperature change could affect the lysosomal membrane integrity differently.

The results from gradual and rapid water temperature changes between 7[degrees]C and 16[degrees]C and between 16[degrees]C and 25[degrees]C showed that in H. rubra the lysosomal membrane stability at the final temperatures of 7[degrees]C, 16[degrees]C, and 25[degrees]C were not significantly affected (P > 0.05) by the rate (gradual or rapid) the water temperature was changed. A similar phenomenon was found by Zhang et al. (2006) in C. gigas at the final temperatures of 5[degrees]C, 15[degrees]C and 25[degrees]C.

In this study a logical correlation between water temperature changes and NRR levels has been established in blacklip abalone, H. rubra. In other publications high water temperature has been documented to affect the growth, health and survival in various abalone species (Prince et al. 1987, Winstanley 1972, Steinbeck et al. 1992, Cheng et al. 2004). It is, therefore, reasonable to expect that by using NRR assay as an indicator, the stress induced by temperature changes in some farming practices could be minimized.

Previous studies also showed that the performances such as growth rate of wild abalone were mainly influenced by temperature, food supply and channeling of energy into gonad production (Ino 1952, Sakai 1962, Poore 1972, Shepherd & Hearn 1983). However, these factors are often highly correlated and it is seldom possible to isolate causal effects between the seasonally changing components (Shepherd & Hearn 1983). It is anticipated that if the impacts of these factors could be quantified by a standard method such as the NRR assay used in this study their relative influences on the performances of wild abalone could then be evaluated. Comparison and prediction of abalone performances at different localities and/or seasons would then become possible. The response of NRR times to temperature changes in blacklip abalone has been characterized in this study. Further work is needed to determine the effects from other factors. Investigations with NRR assay on Pacific oysters have showed that starvation, gonad development and spawning could all individually significantly impair the lysosomal membrane stabilities in blood cells, thus imposing stresses on animals (Cho & Jeong 2005, Zhang & Li 2006, Song et al. submitted).

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TING WANG, (1) XIAOXU LI, (2) * KRISTON BOTT, (2) LIANG SONG,(1) STEVEN CLARKE (2) AND WEN ZHAO (1)

(1) Dalian Fisheries University, Heishijiao, Dalian, People's Republic of China 116023; (2) South Australian Research and Development Institute, 2 Hamra Avenue, West Beach, SA 5024, Australia

* Corresponding author. E-mail: [e-mail]